Insect infestation is responsible for millions of dollars of losses to commercially valuable agricultural crops each year. More than three billion dollars is spent worldwide annually to control insect pests. Traditionally, crops have been controlled from insect pests primarily through the use of toxic sprays. Unfortunately, residues of the sprays remaining on the fruits and vegetables have accumulated in human tissues, often with adverse effects, while at the same time many insects have become immune or resistant to the toxins. Additionally, the sprays often kill useful organisms, and precipitation runoff washes the toxins into streams and other bodies of water often killing fish.
Because of these and other disadvantages of using toxic sprays, alternative means of crop protection have been developed. One approach is the use of biological pesticides. One such agent is the bacteria B. thuringiensis which has been very effective against a variety of caterpillars and worms. This bacterium has been traditionally sold in the form of a dust containing millions of spores. When the spores are sprayed on plants, they are harmless to humans and animals other than the target insect. During its sporulation cycle, Bt produces proteins toxic to certain pests in crystal form known as crystal delta-endotoxins. When the insect ingests any plant tissue with Bt spores on it, Bt .delta.-endotoxin quickly becomes active within the insect's digestive tract, soon paralyzing the gut. The insect stops feeding within two or three hours.
The delta-endotoxin are encoded by crystal protein ("cry") genes. Thus far, over 100 Cry proteins were identified and classified according to their sequence homology and insect specificity (reviewed in Hofte and Whiteley, 1989; Aronson 1993, Schnepf 1995). The cry genes have been divided into six classes and several subclasses based on structural similarities and insecticidal specificity. The major classes are as follows:
______________________________________ Class Insect Specificity ______________________________________ cryI Lepidoptera (butterflies, moths) cryII Lepidoptera and Diptera (flies, mosquitos) cryIII Coleoptera (beetles, weevils) cryIV Diptera (flies, mosquitos) cryV Coleoptera and Lepidoptera cryVI Nematode (roundworms) ______________________________________
With particular regard to the lepidoptera-specific crystal proteins (CryI), to which the present invention is directed, six subclasses having different gene types have been identified. Subclasses of the cryI genes include the following: cryIA (a), cryIA (b), cryIA (c), cryIB, cryIC, and cryID and others. CryIC endotoxin is the most active B. thuringiensis crystal protein against the Spodoptera species which includes the following pests: S. littoralis, S. exempta, S. exigua, S. frugiperda, S. litura and others.
Unfortunately, production of the bacterial spores for commercial use is limited and the protective effect is short-lived. Accordingly, plant molecular biologists have developed transgenic plants that express the Bt toxin within their cells and tissues which have been effective against pests which feed on the leaves of the plant. For example, U.S. Pat. No. 5,187,091 to Donovan et al. describes incorporating into a plant a cryIIIC gene thereby rendering the plant more resistant to insect attack. Additionally, tobacco and tomato plants expressing the Bt toxin gene reportedly have killed larvae of tobacco hornworms. However, the wild-type crystal gene is poorly expressed in transgenic plants. Hence, protection is not attained against less sensitive, but agronomically important, insect pests like the cotton bollworms. (Watson et al. Recombinant DNA, 2d ed. 1992). The expression of the full-length lepidopteran specific Bt gene (cryI) in particular has been reported to be unsuccessful in expressing insecticide in some plants. (Vaeck et al., 1987)
To increase expression in plants, truncated and synthetic genes containing codons preferred in plants have been successfully employed.
U.S. Pat. No. 5,380,831 to Adang et al. discloses a synthetic B. thuringiensis gene designed to be expressed in plants at a level higher than naturally occurring Bt genes. The gene utilizes codons preferred in plants. The modifications described include the elimination of CUUCGG hairpins and plant polyadenylation signals and modifying the A+T content to that found in plants.
U.S. Pat. No. 5,500,365 to Fischoff et al. discloses synthetic plant genes which encode insecticidal proteins of Bt for plant transformation wherein the genes express their protein product at higher levels than the wild-type genes. In particular, they removed regions with many consecutive A+T bases or G+C bases as well as ATTTA sequences and putative polyadenylation signals, and the condon usage was changed according to plant preferences.
The insecticidal spectrum of Bt thus far expressed in transgenic plants is limited. Genes encoding the processed forms of CryIA(a), (b) and (c) have been expressed in plant-associated bacteria and transgenic plants to control major insect pests of maize, rice, cotton, tomato, potato and tobacco. Nonetheless, insects of the Spodoptera species, which cause severe agricultural damage, have thus far escaped efficient control because of problems preventing a high level expression of CryIC toxins in transgenic plants. Therefore, the engineering of Bt toxins with novel specificity is essential for the biological control of recalcitrant plague insects, such as Spodoptera. Members of the Spodoptera genus feed on over 40 different plant families world-wide, including at least 87 species of economic importance. Armyworms, most of which fall within the genus Spodoptera, march in swarms from field to field devastatingly defoliating entire crops. In the United States corn, sorghum and peanut are crops upon which fall armyworm (Spodoptera frugiperda) infestations often reach devastating levels. In one year, for example, losses in the state of Georgia alone were estimated at over 20 million dollars. Corn yield losses attributed to the fall armyworm for the United States have been estimated at 2% annually. In the southeastern United States, S. frugiperda, is a major pest of corn, sorghum and peanut, causing more than $60 million in damages per year. From the various insecticidal crystal proteins of B. thuringiensis expressed in transgenic plants that have been disclosed in prior art none showed activity required for plant protection against Spodoptera species. Moreover, no significant differences in leaf area consumed, mortality or pupal weights of S. exigua larvae were detected between transgenic B. thuringiensis Monsanto cotton line and non-transformed plants (Burns et al., 1994).
The Spodoptera species are polyphagous cutworms and armyworms, that may amplify to enormous numbers and devastate huge agricultural areas. The wide-spread beet armyworm S. exigua attacks rice, sugarbeet, alfalfa, cotton, corn, tobacco, tomato, potato, onions, peas, citrus, sunflower, and many grasses. The Egyptian cotton leafworm S. littoralis,a major pest in African and Mediterranean countries, favors fodder crops, such as alfalfa and clover, but also feeds on many vegetables, industrial crops, medical plants, ornamentals, and trees. Young Spodoptera larvae may be controlled by pyrethroids, DDT, chlorinated hydrocarbons and organophosphorous insecticides. However, because the eggs are laid on grassland, the efficiency of chemical insecticides, including the most efficient compounds methomyl and Pirate (AC303630), is rather limited. During the last decades a considerable effort was therefore invested into the development of safe insecticides to control armyworms in an environmentally friendly fashion.
Despite the significant damage caused by Spodoptera insects, safe and efficient pest control through the genetic engineering of plants is lacking because of the difficulty of achieving a high level of expression of CryIC toxin in transgenic plants. It would therefore be most desirable to have a gene encoding CryIC toxin that can be expressed in transgenic plants thereby safeguarding them against Spodoptera pests in an effective yet environmentally friendly manner.